Thin film bulk acoustic resonator with signal enhancement

ABSTRACT

Sensitivity of thin film bulk acoustic resonance (TFBAR) sensors is enhanced by mass amplification and operating a high frequency.

RELATED APPLICATIONS

This application is a continuation of PCT Patent ApplicationPCT/US2014/027743, filed on Mar. 14, 2014, which claims the benefit ofU.S. Provisional Patent Application No. 61/790,076, filed on Mar. 15,2013. This application also claims the benefit of U.S. ProvisionalPatent Application No. 62/050,589, filed on Sep. 15, 2014. Theabove-referenced applications are hereby incorporated herein byreference in their respective entireties to the extent that they do notconflict with the present disclosure.

FIELD

This disclosure generally relates to, among other things, signalenhancement of thin film bulk acoustic resonators (TFBARs) throughamplification element mediated mass loading.

BACKGROUND

Piezoelectric devices such as thin film bulk acoustic resonators(TFBARs) and similar technologies like quartz crystal microbalances(QCM) have been employed as mass detectors for some time. Oneapplication of piezoelectric resonators is in detecting very smallquantities of materials. Piezoelectric resonators used as sensors insuch applications are sometimes called “micro-balances.” A piezoelectricresonator is typically constructed as a thin, planar layer ofcrystalline or polycrystalline piezoelectric material sandwiched betweentwo electrode layers. When used as a sensor, the resonator is exposed tothe material being detected to allow the material to bind on a surfaceof the resonator.

One conventional way of detecting the amount of the material bound onthe surface of a sensing resonator is to operate the resonator as anoscillator at its resonant frequency. As the material being detectedbinds on the resonator surface, the oscillation frequency of theresonator is reduced. The change in the oscillation frequency of theresonator, presumably caused by the binding of the material on theresonator surface, is measured and used to calculate the amount of thematerial bound on the resonator or the rate at which the materialaccumulates on the resonator surface.

The sensitivity of a piezoelectric resonator in air as a material sensoris theoretically proportional to the square of the resonance frequency.Thus, the sensitivities of material sensors based on the popular quartzcrystal resonators are limited by their relatively low oscillatingfrequencies, which typically range from several MHz to about 100 MHz.The development of thin-film resonator (TFR) technology can potentiallyproduce sensors with significantly improved sensitivities. A thin-filmresonator is formed by depositing a thin film of piezoelectric material,such as AIN or ZnO, on a substrate. Due to the small thickness of thepiezoelectric layer in a thin-film resonator, which is on the order ofseveral microns, the resonant frequency of the thin-film resonator is onthe order of 1 GHz. The high resonant frequencies and the correspondinghigh sensitivities make thin-film resonators useful for material sensingapplications. However, mass sensitivity of even thin-film resonators maybe limited for detection of certain analytes, such as biologicalanalytes.

The use of piezoelectric resonator sensors in immunoassays has beendescribed previously. In general piezoelectric based immunoassays, inwhich mass change is attributable to the immunological reaction betweenan antigen and an antibody, can in circumstances suffer from poorsensitivity and poor detection limit. Consequently, there is a need inthe art for a piezoelectric-based specific binding assay in which thereaction between a molecular recognition component and its targetanalyte can be amplified to provide a more sensitive assay.

One such example is presented in U.S. Pat. No. 4,999,284 issued to Wardon 12 Mar. 1991, which discloses a method using a quartz crystalmicrobalance assay, in which the binding of analyte to a surface on ornear a quartz crystal microbalance (QCM) is detected by a conjugate thatincludes an enzyme. The enzyme is capable of catalyzing the conversionof a substrate to a product capable of accumulating on or reacting witha surface of the QCM leading to a mass change and, hence, a change inresonant frequency.

SUMMARY

This disclosure describes, among other things, signal amplification toenhance sensitivity of TFBAR operating at a high frequency.

In embodiments, a method for detecting an analyte in a sample includescontacting an analyte or an analyte and a tag-linked analyte molecule, afirst recognition component, and a signal amplification element-linkedsecond recognition component to generate a complex comprising the firstrecognition component and the signal amplification element-linked secondrecognition component. The first recognition component is immobilizedrelative to a surface of a thin film bulk acoustic resonator (TFBAR) andis configured to selectively bind one or more of the analyte, theanalyte molecule to which the tag is linked or the tag, or any of thesemolecules that are bound to the second recognition component. The signalamplification element-linked second recognition component is configuredto selectively bind one or more of the analyte, the analyte molecule towhich the tag is linked, or the tag, or any of these molecules that arebound to the first recognition component, or a combination thereof. Themethod further includes contacting the linked signal amplificationelement with one or more amplification precursors under conditions toconvert the precursors into amplification molecules that add mass at asurface of the TFBAR. The added mass may result from deposition of theamplification molecule on the surface; binding of the amplificationmolecule to one or more of the analyte, the tag-linked analyte molecule,the first recognition component or the amplification element-linkedsecond recognition component; or the like. The method also includesobtaining a measure related to the mass (e.g, mass of analyte, signalamplification element-linked second recognition component, andamplification molecule) added at the surface of the TFBAR.

The analyte or the analyte and the tag-linked analyte molecule, thefirst recognition component and the signal amplification element-linkedsecond recognition component may be contacted in any suitable order. Forexample, the analyte or the analyte and the tag-linked analyte moleculemay be contacted with the signal amplification element-linked secondrecognition component prior to contact with the first recognitioncomponent immobilized relative to the surface of the TFBAR. By way offurther example, the analyte or the analyte and the tag-linked analytemolecule may be contacted with the first recognition component prior tocontact with the signal amplification element-linked second recognitioncomponent. By way of yet another example, the analyte or the tag-linkedanalyte molecule, the first recognition component and the signalamplification element-linked second recognition component may becontacted simultaneously.

The signal amplification element may be linked to second recognitioncomponent at any suitable time. In some embodiments, the signalamplification element is linked to the second recognition componentprior to contact with the analyte or the tag-linked analyte molecule. Insome embodiments, the signal amplification element is linked to thesecondary recognition component after the second recognition componentis contacted with the analyte or the tag-linked analyte. In someembodiments, the signal amplification element is linked to the secondrecognition component by a covalent bond. In some embodiments, thesignal amplification element and the second recognition componentinclude moieties that bind with high affinity. By way of example, thesecondary recognition component can be biotinylated, and the signalamplification element may be conjugated to avidin or streptavidin; orvice-versa.

The mass added at the surface of the TFBAR may be measured by anysuitable process. In embodiments, the mass is measured by: (i) couplingan input electrical signal to the TFBAR, the input electric signalhaving a phase and having a frequency within a resonance band of thepiezoelectric resonator, wherein the frequency is about 500 MHz orgreater (such as about 700 MHz or greater, about 800 MHZ or greater,about 900 MHz or greater, about 1 GHz or greater, about 1.2 GHZ orgreater, about 1.4 GHZ or greater, about 1.5 GHZ or greater,1.8 GHZ orgreater, about 2 GHz or greater, about 2.2 GHz or greater, about 2.4 GHzor greater, about 2.5 GHz or greater, from about 500 MHz to about 4 GHz,from about 800 MHz to about 3 GHz, from about 800 MHz to about 10 GHz,or from about 2 GHz to about 2.5 GHz); (ii) transmitting the inputelectrical signal through the TFBAR to generate an output electricalsignal having a frequency and a phase; (iii) receiving the outputelectrical signal from the TFBAR; and (iv) determining a change infrequency or phase of the output electrical signal caused by the addedmass at the surface of the TFBAR, wherein the change in frequency ofphase serves as a measure of the mass added at the surface of the TFBAR.

One or more embodiments of the apparatuses, systems or methods describedherein provide one or more advantages over prior sensors, devices,systems or methods for detecting small quantities of an analyte. Asdescribed herein, at higher frequencies larger TFBAR signalamplification was surprisingly observed with amplificationelement-mediated mass loading than at lower frequencies. Accordingly,advantages of higher frequencies appear to be even further enhanced whenemployed in combination with signal amplification. This and otheradvantages will be readily understood by those of skill in the art fromthe following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C are schematic diagrams illustrating the operationalprinciples of embodiments of thin film bulk acoustic resonator (TFBAR)sensing devices.

FIG. 2 is a schematic diagram showing components of a TFBAR system fordetecting an analyte.

FIGS. 3A-D are schematic drawings illustrating an embodiment of signalamplification on a surface of a thin film resonator (TFR).

FIGS. 4A-D are schematic drawings illustrating an embodiment of signalamplification on a surface of a TFR.

FIGS. 5A-B are schematic diagrams of an embodiment of a first bindingpartner bound to a surface of a TFR (5A) and a recognition componentbound to a second binding partner, which is bound to the first bindingpartner (5B).

FIG. 6A is a plot of response over time of direct analyte binding andenzyme amplified analyte binding on and embodiment of a TFBAR.

FIG. 6B is a plot showing details of a portion of the plot presented inFIG. 4A.

FIG. 7 is a schematic drawing illustrating an embodiment of variouspolynucleotide components bound to a surface of a TFR as described inEXAMPLE 2.

The schematic drawings are not necessarily to scale. Like numbers usedin the figures refer to like components, steps and the like. However, itwill be understood that the use of a number to refer to a component in agiven figure is not intended to limit the component in another figurelabeled with the same number. In addition, the use of different numbersto refer to components is not intended to indicate that the differentnumbered components cannot be the same or similar.

DETAILED DESCRIPTION

In the following detailed description several specific embodiments ofcompounds, compositions, products and methods are disclosed. It is to beunderstood that other embodiments are contemplated and may be madewithout departing from the scope or spirit of the present disclosure.The following detailed description, therefore, is not to be taken in alimiting sense.

This disclosure generally relates to, among other things, methods,devices, sensors and systems for detecting an analyte. The method,devices, sensors and systems use a thin film bulk acoustic resonator(TFBAR) that measures a change in frequency or phase of the resonatorcaused by the binding of the analyte on a surface of the resonator. Thebinding signal is enhanced through amplification element-mediated massloading. An input electrical signal having a phase and having afrequency within a resonance band of the piezoelectric resonator, whichin the case of some embodiments of the present disclosure may be about500 MHz or greater, such as about 1.5 GHz or greater, is coupled to andtransmitted through the resonator to generate an output electricalsignal which is frequency-shifted or phase-shifted from the input signaldue to binding, deposition, etc. of material being detected on theresonator surface and amplification due to the amplificationelement-mediated mass loading. The output electrical signal receivedfrom the piezoelectric resonator is analyzed to determine the change infrequency or phase caused by the binding of analyte and amplificationelement mediated mass deposition on the resonator surface. The measuredchange in frequency or phase provides quantitative information regardingthe analyte (or tag-linked analyte molecule) bound to the resonatorsurface.

Sensors, Devices and Systems

The sensors disclosed herein include at least one thin film resonatorsensor, such as a thin film bulk acoustic resonator (TFBAR) sensor. ATFBAR sensor includes a piezoelectric layer, or piezoelectric substrate,and input and output transducer. TFBAR sensors are small sensors makingthe technology suitable for use in handheld devices. Accordingly, ahandheld device for detecting target analytes comprising a sensordescribed herein is contemplated.

Turning now to the drawings with reference to FIGS. 1A and 1B, generaloperating principles of an embodiment of a bulk-acoustic wavepiezoelectric resonator 20 used as a sensor to detect an analyte areshown. The resonator 20 typically includes a planar layer ofpiezoelectric material bounded on opposite sides by two respective metallayers which form the electrodes of the resonator. The two surfaces ofthe resonator are free to undergo vibrational movement when theresonator is driven by a signal within the resonance band of theresonator. When the resonator is used as a sensor, at least one of itssurfaces is adapted to provide binding sites for the material beingdetected. The binding of the material on the surface of the resonatoralters the resonant characteristics of the resonator, and the changes inthe resonant characteristics are detected and interpreted to providequantitative information regarding the material being detected.

By way of example, such quantitative information may be obtained bydetecting a change in the insertion or reflection coefficient phaseshift of the resonator caused by the binding of the material beingdetected on the surface of the resonator. Such sensors differ from thosethat operate the resonator as an oscillator and monitor changes in theoscillation frequency. Rather such sensors insert the resonator in thepath of a signal of a pre-selected frequency and monitor the variationof the insertion or reflection coefficient phase shift caused by thebinding of the material being detected on the resonator surface. Ofcourse, sensors that monitor changes in oscillation frequency may alsobe employed in accordance with signal amplification described herein.

In more detail, FIG. 1A shows the resonator 20 before the material beingdetected is bound to its surface 26. The depicted resonator 20 iselectrically coupled to a signal source 22, which provides an inputelectrical signal 21 having a frequency f within the resonance band ofthe resonator. The input electrical signal is coupled to the resonator20 and transmitted through the resonator to provide an output electricalsignal 23. In the depicted embodiment, the output electrical signal 23is at the same frequency as the input signal 21, but differs in phasefrom the input signal by a phase shift ΔΦ₁, which depends on thepiezoelectric properties and physical dimensions of the resonator. Theoutput signal 23 is coupled to a phase detector 24 which provides aphase signal related to the insertion phase shift.

FIG. 1B shows the sensing resonator 20 with the material being detectedbound on its surface 26. The same input signal is coupled to theresonator 20. Because the resonant characteristics of the resonator arealtered by the binding of the material as a perturbation, the insertionphase shift of the output signal 25 is changed to ΔΦ₂. The change ininsertion phase shift caused by the binding of the material is detectedby the phase detector 24. The measured phase shift change is related tothe amount of the material bound on the surface of the resonator.

FIG. 1C shows an alternative to measuring the insertion phase of theresonator. A directional coupler 27 is added between the signal source22 and the resonator 20 with the opposite electrode grounded. A phasedetector 28 is configured to measure the phase shift of the reflectioncoefficient as a result of material binding to the resonator surface.

Other TFBAR phase-shift sensors that may be employed with the signalamplification aspects described herein include those described in, forexample, U.S. Pat. No. 8,409,875 entitled “RESONATOR OPERTING FREQUENCYOPTIMIZATION FOR PHASE-SHIFT DETECTION SENSORS,” which patent is herebyincorporated herein by reference in its entirety to the extent that itdoes not conflict with the disclosure presented herein. For example,sensor apparatuses may include (i) a sensing resonator comprisingbinding sites for an analyte; (ii) actuation circuitry configured todrive the sensing resonator in an oscillating motion; (iii) measurementcircuitry arranged to be coupled to the sensing resonator and configuredto measure one or more resonator output signals representing resonancecharacteristics of the oscillating motion of the sensing resonator; and(iv) a controller operatively coupled with the actuation and measurementcircuitry. The controller can be interfaced with data storage containinginstructions that, when executed, cause the controller to adjust thefrequency at which the actuation circuitry drives the sensing resonatorto maintain a resonance point of the sensing resonator. Accordingly,sensing may be accomplished by actuating the TFBAR into an oscillatingmotion; measuring one or more resonator output signals representingresonance characteristics of the oscillating motion of the TFBAR; andadjusting the actuation frequency of the sensing resonator to maintain aresonance point of the TFBAR. In embodiments, the frequency at which theactuation circuitry drives the sensing resonator is a frequency ofmaximum group delay.

Such phase detection approaches can be advantageously used withpiezoelectric resonators of different resonant frequencies.

In various embodiments, TFBARs for use with the methods, devices, andsystem described herein have resonance frequencies of about 500 MHz orgreater, such as about 700 MHz or greater, about 900 MHz or greater,about 1 MHz or greater, 1.5 GHz or greater, about 1.8 GH or greater,about 2 GHz or greater, 2.2 GHz or greater, 2.5 GHz or greater, about 3GHZ or greater, or about 5 GHZ or greater can provide enhancedsensitivity when used with amplification element mediated mass loaded,which is described in more detail below. In embodiments, the TFBARs haveresonance frequencies of from about 500 MHz to about 5 GHz, such as fromabout 900 MHz to about 3 GHz, or from about 1.5 GHz to about 2.5 GHz.Some of such frequencies are substantially higher than frequencies ofpreviously described piezoelectric resonators.

The sensing resonators described herein are thin-film resonators. Thinfilm resonators comprise a thin layer of piezoelectric materialdeposited on a substrate, rather than using, for example, AT-cut quartz.The piezoelectric films typically have a thickness of less than about 5micrometers, such as less than about 2 micrometers, and may havethicknesses of less than about 100 nanometers. Thin-film resonators aregenerally preferred because of their high resonance frequencies and thetheoretically higher sensitivities. Depending on the applications, athin-film resonator used as the sensing element may be formed to supporteither longitudinal or shear bulk-acoustic wave resonant modes.Preferably, the sensing element is formed to support shear bulk-acousticwave resonant modes, as they are more suitable for use in a liquidsample.

Additional details regarding sensor devices and systems that may employTFRs are described in, for example, U.S. Pat. No. 5,932,953 issued Aug.3, 1999 to Drees et al., which patent is hereby incorporated herein byreference in its entirety to the extent that it does not conflict withthe disclosure presented herein.

TFR sensors may be made in any suitable manner and of any suitablematerial. By way of example, a resonator may include a substrate such asa silicon wafer or sapphire, a Bragg mirror layer or other suitableacoustic isolation means, a bottom electrode, a piezoelectric material,and a top electrode.

Any suitable piezoelectric material may be used in a TFR. Examples ofsuitable piezoelectric substrates include lithium tantalate (LiTaO₃),lithium niobate (LiNbO₃), Zinc Oxide (ZnO), aluminum nitride (AlN),plumbum zirconate titanate (PZT) and the like.

Electrodes may be formed of any suitable material, such as aluminum,tungsten, gold, titanium, molybdenum, or the like. Electrodes may bedeposited by vapor deposition or may be formed by any other suitableprocess.

Any suitable device or system may employ a thin film resonator andamplification as described herein. By way of example and with referenceto FIG. 2, a system for detecting an analyte may include a container 10(or more than one container), the thin film resonator 20, actuationcircuitry 22, measurement circuitry 29, and control electronics 30. Afluid path couples the one or more containers 10 to the resonator 20.The control electronics 30 are operably coupled to the actuationcircuitry and the measurement circuitry. In embodiments, controlelectronics 30 are configured to modify the frequency at which theactuation circuitry 22 oscillates the resonator 20 based on input fromthe measurement circuitry 29.

Still with reference to FIG. 2, the container 10 (or more than onecontainer) may house an amplification molecule, an amplificationelement-linked second recognition component or components thereof, andoptionally one or more of a tag, an analyte molecule, and a firstrecognition component. Each of these reagents is described in moredetail below. Control electronics 30 may control the flow of suchreagents from container 10 to resonator 20; e.g. via a pump, vacuum, orthe like.

Any suitable control electronics 30 may be employed. For example,control electronics may include a processor, controller, memory, or thelike. Memory may include computer-readable instructions that, whenexecuted by processor or controller cause the device and controlelectronics to perform various functions attributed to device andcontrol electronics described herein. Memory may include any volatile,non-volatile, magnetic, optical, or electrical media, such as a randomaccess memory (RAM), read-only memory (ROM), non-volatile RAM (NVRAM),electrically-erasable programmable ROM (EEPROM), flash memory, or anyother digital media. Control electronics 30 may include any one or moreof a microprocessor, a controller, a digital signal processor (DSP), anapplication specific integrated circuit (ASIC), a field-programmablegate array (FPGA), or equivalent discrete or integrated logic circuitry.In some examples, control electronics 30 may include multiplecomponents, such as any combination of one or more microprocessors, oneor more controllers, one or more DSPs, one or more ASICs, or one or moreFPGAs, as well as other discrete or integrated logic circuitry. Thefunctions attributed to control electronics herein may be embodied assoftware, firmware, hardware or any combination thereof.

Molecular Recognition and Signal Amplification

Molecular recognition of a sample comprising a significant backgroundsignal may be facilitated by amplification of the signal. The sensors,systems and methods described herein employ a second recognitioncomponent comprising an amplification element such as a linked enzyme.The TFBAR sensors, at the higher frequency ranges described herein,responded very efficiently to mass increase of the sensor surface due toprecipitation of a substrate cleaved by an enzyme.

Referring now to FIGS. 3A-D, schematic drawings illustration enzymeamplification on a TFBAR are shown. As depicted in FIG. 3A, a molecularrecognition component 100 configured to bind to an analyte isimmobilized on a surface 26 of a resonator 20. The resonator 20 havingimmobilized molecular recognition component 100 may be contacted with acomposition comprising an analyte 110, which may bind molecularrecognition component 100 (see FIG. 3B). The resonator 20 havingimmobilized molecular recognition component 100 to which analyte 110 isbound may be contacted with a composition comprising a second molecularrecognition component 120 linked to an amplification element 130 such asan enzyme. The second molecular recognition component 120 is configuredto bind to analyte 110 such that the second molecular recognitioncomponent 120 and linked amplification element 130 are immobilizedrelative to the surface 26 (see FIG. 3C). In the depicted embodiments, asoluble substrate 140 may be converted by amplification element 130 toan insoluble product 150, which precipitates and accumulates on thesurface 26 of the sensor 20, thereby amplifying the mass signal as afunction of amount or concentration of bound analyte 110 (see FIG. 3D).

It will be understood that the series of events depicted in FIGS. 3A-3Dare shown for purposes of illustration and that any other suitablesequence of events may be employed. For example, the analyte 110 may becontacted with the second molecular recognition component 120 (and boundamplification element 130) before the analyte (with bound secondmolecular recognition component) is contacted to the surface 26 of theresonator 20 relative to which the molecular recognition component 100is immobilized. The substrate 140 may be present at the time the secondmolecular recognition component 120—amplification element 130 is addedor may be added later. In any case, washing may be performed prior toamplification.

Non-limiting examples of target analytes include nucleic acids,proteins, peptides, antibodies, enzymes, carbohydrates, chemicalcompounds, or infectious species such as bacteria, fungi, protozoa,viruses and the like. In certain applications, the target analyte iscapable of binding more than one molecular recognition component.

Any suitable molecular recognition component (e.g., 100 in FIG. 3) maybe bound to the surface of a resonator. The molecular recognitioncomponent preferably selectively binds to the analyte of interest. Byway of example, the molecular recognition component may be selected fromthe group consisting of nucleic acids, nucleotide, nucleoside, nucleicacids analogues such as PNA and LNA molecules, proteins, peptides,antibodies including IgA, IgG, IgM, IgE, lectins, enzymes, enzymescofactors, enzyme substrates, enzymes inhibitors, receptors, ligands,kinases, Protein A, Poly U, Poly A, Poly lysine, triazine dye, boronicacid, thiol, heparin, polysaccharides, coomassie blue, azure A,metal-binding peptides, sugar, carbohydrate, chelating agents,prokaryotic cells and eukaryotic cells.

Any suitable method for immobilizing a molecular recognition componenton a surface of a TFBAR may be used. By way of example, a uniformcoating of epoxy silane may be deposited on the sensor surface using avapor deposition process. Test and reference molecular recognitioncomponents, such as antibodies, may then be deposited onto the test andreference resonators using, for example, piezo based nanodispensingtechnology. Primary amines on the antibodies react with the epoxidegroups covalently binding the antibody to the sensor surface. By way offurther example, a thiol group, if present, of the molecular recognitioncomponent bind to a surface of the TFBAR. The surface of the TFBAR maybe modified, as appropriate or necessary, to permit binding of themolecular recognition component.

Any suitable molecular recognition components, such as those describedabove, may be used as the second molecular recognition component (e.g.,120 in FIG. 3). The second molecular recognition component may be linkedto any suitable amplification element, such as an enzyme. Preferably,the second molecular recognition component is an antibody and theamplification element is an enzyme.

Any suitable amplification element may be linked to the second molecularrecognition component. In embodiments, the amplification element is anactivatable polymerization initiator, such as a photoinitiator, achemical initiator, or a thermoinitiator. The polymerization initiatormay be activated in the presence of one or more monomers to cause apolymer to graft from the second molecular recognition component. Inembodiments, the amplification element is an enzyme. In embodiments, theenzyme is capable of converting a substrate that is soluble in the assayenvironment to an insoluble product that precipitates on the surface ofthe sensor. Examples of suitable enzymes include alkaline phosphatase(ALP), horse radish peroxidase (HRP), beta galactosidase, and glucoseoxidase.

Examples of enzyme/substrate systems that are capable of producing aninsoluble product which is capable of accumulating on a surface of aTFBAR include alkaline phosphatase and5-bromo-4-chloro-3-indolylphosphate/nitro-blue tetrazolium chloride(BCIP/NBT). The enzymatically catalyzed hydrolysis of BCIP produces aninsoluble dimer, which may precipitate on the surface of the sensors.Other analogous substrates having the phosphate moiety replaced withsuch hydrolytically cleavable functionalities as galactose, glucose,fatty acids, fatty acid esters and amino acids can be used with theircomplementary enzymes. Other enzyme/substrate systems include peroxidaseenzymes, for example horse radish peroxidase (HRP) or myeloperoxidase,and one of the following: benzidene, benzidene dihydrochloride,diaminobenzidene, o-tolidene, o-dianisidine and tetramethyl-benzidene,carbazoles, particularly 3-amino-9-ethylcarbazole, and various phenoliccompounds all of which have been reported to form precipitates uponreaction with peroxidases. Also, oxidases such as alphahydroxy acidoxidase, aldehyde oxidase, glucose oxidase, L-amino acid oxidase andxanthine oxidase can be used with oxidizable substrate systems such as aphenazine methosulfate-nitriblue tetrazolium mixture.

It will be understood that any type of competition assay may beemployed. It will be further understood that the analyte may be modifiedto include a tag recognizable by the first or second recognitioncomplex, such as a streptavidin tag; biotin tag; a chitin bindingprotein tag; a maltose binding protein tag; a glutathione-S-transferasetag; a poly(His) tag; an epitope tag such as a Myc tag, a HA tag, or aV5 tag; or the like. It will be further understood that the tag-linkedanalyte may include a variant or derivative of the analyte. The variantor derivative is a variant or derivative that is selectivelyrecognizable by the first or second molecular recognition component thatis configured to recognize the analyte. In some situations, it may bedesirable that the variant or derivative analyte have an affinity forthe first or second molecular recognition component that is differentthan the affinity of the non-tag-linked analyte. The variant orderivative of the analyte may be a variant or derivative that allows forease of manufacture of the tag-linked analyte. For example, thetag-linked analyte may comprise a recombinant polypeptide, etc.

When competition assays employing tag-linked analyte molecules areperformed, the tag-linked analyte molecule, rather than or in additionto the analyte, may bind a first molecular recognition componentimmobilized on a surface of a resonator.

Referring now to FIGS. 4, an embodiment of a signal amplification assayis depicted. Many of the components of FIG. 4 are the same or similar tothe components depicted in FIG. 3. If a particular element is notspecifically discussed with regard to FIG. 4, reference is made to thenumbered element in FIG. 3 above. A signal amplification element 130 maybe linked to second recognition component 120 at any suitable time. Insome embodiments (not depicted in FIG. 4), the signal amplificationelement 130 is linked to the second recognition component 120 prior tocontact with the analyte 110 or the tag-linked analyte molecule. In someof such embodiments, the signal amplification element 130 is covalentlybound to the second recognition component 120.

In some embodiments (e.g., as depicted in FIGS. 4C-D), signalamplification element 130 is linked to secondary recognition component120 after second recognition component 120 is contacted with the analyte110 or the tag-linked analyte. By way of example, second recognitioncomponent 120 can include first binding partner 123 configured toselectively bind second binding partner of signal amplification element130. First binding partner 123 is preferably covalently bound to secondrecognition component 120. Second binding partner 135 is preferablycovalently bound to signal amplification element 130. First 123 andsecond 135 binding partners preferably bind with high affinity.

Any suitable combination of first 123 and second 135 binding partnersmay be employed. By way of example, secondary recognition component 120can be biotinylated, and signal amplification element 130 may beconjugated to streptavidin; or vice-versa. By way of further example,one of first and second binding partners can be a polyhistidine(His)tag, and the other of first and second binding partners can be, forexample, a nickel or copper chelator, such as as iminodiacetic acid(Ni-IDA) and nitrilotriacetic acid (Ni-NTA) for nickel andcarboxylmethylaspartate (Co-CMA) for cobalt, which the poly(His) tag canbind with micromolar affinity. Generally nickel-based resins have higherbinding capacity, while cobalt-based resins offer the highest purity. Byway of yet another example, one of first and second binding partners canbe a glutathione-S-transferase (GST) tag, and the other of first andsecond binding partners can be glutathione. For yet another example, oneof first and second binding partners can be a maltose binding proteintag, and the other of first and second binding partners can be amyloseor maltose. As another example, one of first and second binding partnerscan be a chitin binding protein tag, and the other of first and secondbinding partners can be chitin. It will be understood thatabove-presented binding partners are merely examples of high affinitybinding partners that may be conjugated to a second recognitioncomponent or a signal amplification element and that other bindingpartners are contemplated herein. Further, it will be understood thatmore than one set of binding partners may be employed to link secondrecognition component to signal amplification element.

Binding partners may be conjugated to second recognition component orsignal amplification element through any suitable technique. Forexample, chemical conjugation or recombinant techniques may be employedto link a binding partner to second recognition component or signalamplification element, as appropriate. Such techniques are well known tothose of skill in the art. For example, a heterobifunctional crosslinker utilizing NHS-ester and maleimide functional groups may beemployed as known to those of skill in the art.

It will be understood that, if signal amplification element and secondrecognition component include complementary binding partners, the signalamplification element may be linked to second recognition component viabinding partners at any suitable time. For example and as shown in FIGS.4C-D, signal amplification element 130 may be linked to secondrecognition component 120 after second recognition component 120 iscontacted with analyte 110 or tag-linked analyte. In some embodiments,signal amplification element 130 containing second binding partner 135is contacted with second recognition component 120 containing firstbinding partner 123 before second recognition component 120 is contactedwith analyte 110 or tag-linked analyte or at the same time as secondrecognition component 120 is introduced to resonator.

Referring now to FIGS. 5A-B, a first molecular recognition component 100may be bound to a surface 26 of a TFBAR 20 via one or more intermediate.For example, a first binding partner 99 may be bound to the surface 26and the first molecular recognition component 100 may include a secondbinding partner 101 configured to selectively bind to the first bindingpartner 99. The binding partners 99, 101 may be binding partners asdescribed above (e.g., with regard to FIG. 4). First recognitioncomponent 100 may be bound to surface 26 via binding partners 99, 101 atany suitable time, such as before the sensor 20 is incorporated into adevice or system or after the sensor 20 is incorporated into the deviceor system. For example, first recognition component 100 may be bound tosurface 26 via binding partners 99, 101 as a first step of, or during,an analyte detection assay.

Amplification Element-Mediated Mass Loading/Signal Amplification withTFBARs

It has been noted that, as resonance frequency increases, sensitivityfor mass detection should also increase. However, this is not alwaysobserved in practice. In theory, a TFBAR having a resonance frequency toabout 2.2 GHz should afford sufficient sensitivity to detect lowconcentrations of analytes without the use of signalamplification/mass-loading as described herein. However, the inventorsfound that even with such high resonance frequencies, TFBAR sensors werenot sufficiently sensitive to detect low levels of analyte. However, byutilizing the amplification/mass-loading techniques described herein,more of the theoretical gains in sensitivity offered by operating athigher frequencies can be realized.

Susceptibility to noise is related to signal propagation discussedtheoretically above. At higher frequencies the signal propagates shorterdistances, thus creating a proximity filter. That is, you only measurewhat is in proximity to the surface. However, what constitutes proximitywill change with frequency and can have important practicalramifications with regard to susceptibility to background noise.Operation at higher frequencies with the mass loading not only resultsin enhanced signal sensitivity, it also results in lower susceptibilityto noise. That can translate functionally to, for example, lessstringent washing requirements because the amplifier linked secondmolecular recognition component that is not bound to the surface of theresonator (e.g., via analyte bound to first molecular recognitioncomponent) should not add significant mass in proximity to the surfaceof the resonator. Furthermore, the washing requirements to obtain astable baseline reading in negative sample were found to be much lessstringent with higher frequency TFBARs, which may also be due to theshorter distance of signal propagation at higher frequency.

Surprisingly, it has been found that larger signal amplification isobserved at higher frequencies than at lower frequencies. See, e.g.Table 2 in the EXAMPLES below, where larger signal amplification wasobserved with enzyme mediated mass loading (relative to direct binding)at 2250 MHz compared with 900 MHz resonators. That larger levels ofsignal amplification can be obtained at higher frequencies wasunexpected because the different resonators (900 MHz and 2250 MHz) wereconstructed to contain the same concentration or amount of firstrecognition component, assays used the same concentration and amount ofanalyte, and the same concentration and amount of enzyme-linked secondmolecular recognition component and substrate were used. Thus, intheory, the amount of amplification that actually occurs would beexpected to be the same (the amount of product precipitated on thesurface would be expected the same). However, a larger amount ofamplification of signal was observed at higher frequencies.

Use

The sensors, devices and systems described herein may be employed todetect an analyte in a sample. The sensors may find use in numerouschemical, environmental, food safety, or medial applications. By way ofexample, a sample to be tested may be, or may be derived from blood,serum, plasma, cerebrospinal fluid, saliva, urine, and the like. Othertest compositions that are not fluid compositions may be dissolved orsuspended in an appropriate solution or solvent for analysis.

Definitions

All scientific and technical terms used herein have meanings commonlyused in the art unless otherwise specified. The definitions providedherein are to facilitate understanding of certain terms used frequentlyherein and are not meant to limit the scope of the present disclosure.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” encompass embodiments having pluralreferents, unless the content clearly dictates otherwise.

As used in this specification and the appended claims, the term “or” isgenerally employed in its sense including “and/or” unless the contentclearly dictates otherwise. The term “and/or” means one or all of thelisted elements or a combination of any two or more of the listedelements.

As used herein, “have”, “having”, “include”, “including”, “comprise”,“comprising” or the like are used in their open ended sense, andgenerally mean “including, but not limited to”. It will be understoodthat “consisting essentially of”, “consisting of”, and the like aresubsumed in “comprising” and the like. As used herein, “consistingessentially of,” as it relates to a composition, product, method or thelike, means that the components of the composition, product, method orthe like are limited to the enumerated components and any othercomponents that do not materially affect the basic and novelcharacteristic(s) of the composition, product, method or the like.

The words “preferred” and “preferably” refer to embodiments of theinvention that may afford certain benefits, under certain circumstances.However, other embodiments may also be preferred, under the same orother circumstances. Furthermore, the recitation of one or morepreferred embodiments does not imply that other embodiments are notuseful, and is not intended to exclude other embodiments from the scopeof the disclosure, including the claims.

Also herein, the recitations of numerical ranges by endpoints includeall numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2,2.75, 3, 3.80, 4, 5, etc. or 10 or less includes 10, 9.4, 7.6, 5, 4.3,2.9, 1.62, 0.3, etc.). Where a range of values is “up to” a particularvalue, that value is included within the range.

Any direction referred to herein, such as “top,” “bottom,” “left,”“right,” “upper,” “lower,” and other directions and orientations aredescribed herein for clarity in reference to the figures and are not tobe limiting of an actual device or system or use of the device orsystem. Devices or systems as described herein may be used in a numberof directions and orientations.

“Binding event,” as used herein, means the binding of a target analyteto a molecular recognition component immobilized in a surface of asensor.

EXAMPLES

The following non-limiting examples serve to describe more fully themanner of using the above described sensors, methods, devices andsystems. It is understood that these examples in no way serve to limitthe scope of this disclosure or claims that follow, but rather arepresented for illustrative purposes.

Example 1 Enzyme Amplification Proof of Concept

Initial proof of concept studies were performed using an anti-bovine IgGassay and alkaline phosphatase (ALP) as the conjugated enzyme withBCIP/NBT as the precipitating substrate. Briefly, a goat anti-bovine andgoat anti-rat antibodies were immobilized on the test and referenceresonators by dispensing 350 μm spots using a piezo dispenser onto epoxysilane coated sensors with a resonate frequency of 2.2 GHz. Sensors wereincubated overnight in a high humidity environment at 4° C. Sensors wereblocked with fish skin gelatin prior to testing. The reference signalwas then subtracted from the test signal and this delta signal used asthe binding response. All testing performed with the sensors immersed inmicrotiter plates. Sample agitation achieved by using stirbars. Thetesting sequence was as follows, sensors were exposed to 1 μg/ml BovineIgG for 60 seconds followed by a 30 second rinse and exposure to arabbit anti-bovine IgG-alkaline phosphatase conjugate for 60 seconds.Sensors then rinsed 2 times for 30 seconds and exposed to BCIP/NBTsubstrate for 60 seconds. Sensors electrically connected to a networkanalyzer which was used to monitor the frequency shift of the devices.In this case, phase resulting in maximum group delay was tracked andchange in input frequency to maintain the phase as mass changed wasdetermined. A 50 MHz window around the resonate frequency was collect ata sampling rate of 2 samples per second for both the test and referenceresonators. This data was post processed to determine the frequencyshift as a function of time for both the test and reference resonators.The frequency shift observed from direct antigen binding was thencompared to the signal observed in the enzyme substrate.

Results of this initial study are presented in FIG. 4A, with FIG. 4Bbeing a detailed view of a portion of the plot presented in FIG. 4A. Asshown, the ALP-enzyme amplification resulted in significant improvementin sensitivity relative to direct binding without addition of thesubstrate. As shown in Table 1 below, over 100 fold amplification inresponse and slope was observed as a result of enzyme amplification.

TABLE 1 Results of ALP-Amplification Direct Enzyme Binding AmplificationResponse (Integral) −9,024 −1,094,473 Amplification (X) 121 Slope −3.24−374.77 Slope Amp 115.71

Similar studies were performed using anti-bovine IgG assay and horseradish peroxidase (HRP) as the conjugated enzyme. The precipitatingsubstrate in this reaction was hydrogen peroxide and p-hydroxycinnamicacid (data not shown). Further analysis on the ALP enhancement withBCIP/NBT using an anti-rat IgG assay was also performed (data notshown). Subsequent development work with the ALP and BCIP/NBT system hasincluded evaluation with a variety of other.

The benefits of precipitate amplification appear to be frequencydependent. Devices with operating frequencies of 2250 MHz and 850 MHzwere coated with a Goat anti-Rat F(ab′) fragment. The native sulfhydrylgroup on the reduced F(ab′) was used to link to the gold resonatorsurface forming a dative sulfur gold bond. These sensors were thenblocked with fish skin gelatin and tested in negative buffer sample or 1μg/ml Rat IgG followed by incubation with a goat anti-Rat alkalinephosphatase conjugated antibody. Sensors were then rinsed two times andexposed to BCIP/NBT substrate. Data was reduced as previously describedfor both the direct binding event and the substrate amplification.Comparison of the 2250 MHz to 900 MHz TFBARs demonstrate a 2.5 foldincrease in signal amplification with the higher frequency (2250 MHz)relative to the 850 MHz devices. Additionally, the level of backgroundobserved in negative sample was considerably higher in the 850 MHzdevices. This was true when the data was analyzed as kHz/sec of responsebut the difference became even more dramatic when the results wereconverted to ppm/sec by dividing the frequency shift response by thefrequency of operation. The addition of two extra wash steps prior tosubstrate exposure reduced the amount of background signal in the 850MHz devices to levels comparable to those seen in the 2250 MHz devices(data not shown).

TABLE 2 Comparison of amplification with 850 and 2250 MHz TFBARFrequency (MHz) 2250 850 Background Signal in Negative (ppm/sec) −0.86−23.11 Direct Binding (kHz/sec) −3.26 −0.70 Amplified signal (kHz/sec)−233.7 −22.3 Amplification (X) 71.7 31.9

Example 2 DNA Proof of Feasibility

For DNA binding to the sensors surface a 10 μM 5′-amine labeled 27-mer(complementary to the 3′ end of the target oligonucleotide) and 10 μM ofa missense 27-mer were both dissolved in 3× saline-sodium citrate (3×SSC) and spotted on epoxy silane functionalized sensors as the test andreference respectively (2150 MHz TFBAR).

A 125-mer oligonucleotide was used as a model target. The targetoligonucleotide was mixed with 6 nM of a 3′biotin labeled 18-mercomplementary to the 5′ end of the 125-mer target in hybridizationbuffer (5×SSC, 10% formamide, 0.1% SDS) and reacted with the sensorsurface for 4 minutes at 39 C.

A schematic diagram of the 27-mer bound to the sensor surface and thetarget 125-mer, which is bound to the biotinylated 18-mer is shown inFIG. 7.

Two wash steps were then performed (1×SSC with 0.01% SDS and HEPESbuffered saline plus detergent) and the sensor was exposed to 2 μg/mlstreptavidin-alkaline phosphatase conjugate purchased from Jackson Immuo(Part Number 016-050-084) for 2 minutes in HEPES buffer containing 1mg/ml fish skin gelatin (FSG).

Two additional wash steps were performed with HEPES plus detergent andthe sensor was exposed to the precipitating substrate NBT/BCIP purchasedfrom Thermo (Part Number 34042). Frequency shift data was collected for1.5 minutes at 39 C and is presented below in Table 3. Estimated limitof detection using the zero response plus 3 standard deviations yieldeda value of 0.6 pM.

TABLE 3 Frequency Shift Data DNA Target Average Response (pM) (kHz/sec)0 0.93 0.8 −0.48 4 −2.93 40 −38.15 400 −111.61

Example 3 Interleukin-6 (IL-6) Two-Step Immunoassay

Reagents:

Affinity purified goat anti IL-6 (R&D Systems Part Number AF-206-NA) wasspotted onto epoxy silane activated sensors (2175 MHz TFBAR) generallyas discussed above for the antibody in EXAMPLE 1.

Mouse monoclonal antibody against IL-6 (R&D Systems Part Number MAB206)was labeled with a 5× molar excess of sulfo-NHS-LC-Biotin (ThermoScientific) according to the manufacturer's instructions. Excessunincorporated biotin reagent was removed by desalting.

Calibrator matrix was prepared by mixing 10% (v/v) chicken serum(Equitech, charcoal stripped, heat inactivated) with phosphate bufferedsaline (PBS) plus 0.1% sodium azide. Calibrators were prepared bydiluting recombinant human IL-6 (R&D Systems Part Number 206-IL) inCalibrator matrix.

Streptavidin conjugated to alkaline phosphatase (SA-ALP) was purchasedfrom Jackson Immuno (Part Number 016-050-084).

Wash buffer is Hepes buffered saline plus detergent.

Substrate is 1-Step NBT/BCIP purchased from Thermo Scientific (PartNumber 34042).

Assay:

Biotinylated mouse anti IL-6 was diluted to a working strength of 8.3μg/mL in a Hepes buffer containing fish skin gelatin (Hepes/FSG).

SA-ALP was diluted to a working strength of 1 μg/mL in Hepes/FSG.

60 μL of biotinylated mouse anti IL-6 was mixed with 40 μL of calibratorand passed back and forth over the sensor for 16 minutes. The reactionmixture was then removed from the microfluidic channel and SA-ALP waspassed back and forth over the sensor for 2 minutes. The sensor was thenwashed three times with Wash buffer followed by addition of BCIP/NBTsubstrate. The rate of change in resonance frequency during thesubstrate step was plotted with respect to IL-6 concentration. Theresults are shown in the Table 4 below including an estimate of theanalytical sensitivity (zero response plus 3 standard deviations).

TABLE 4 Frequency Shift for IL-6 Assay Response Calcu- % IL6 (kHz/sec)lated Resid- Re- (pg/mL) Run Avg StDev CV (pg/mL) uals covery 0 −2.28−2.12 0.23 11% 0.83 0.83 −1.96 <0.83 NA NA 5 −3.53 −3.48 0.08  2% 5.830.83 116.6 −3.42 5.42 0.42 108.4 10 −3.75 −4.67 0.88 19% 6.65 −3.35 66.5−4.75 10.23 0.23 102.3 −5.51 12.87 2.87 128.7 25 −8.61 −9.24 0.89 10%23.32 −1.68 93.28 −9.87 27.48 2.48 109.92 100 −28.49 −31.44 2.77  9%90.05 −9.95 90.05 −33.99 109.87 9.87 109.87 −31.85 102.05 2.05 102.050 + −2.82 analytical 3.11 3SD sensitivity estimate

Thus, embodiments of THIN FILM BULK ACOUSTIC RESONATOR WITH SIGNALENHANCEMENT are disclosed. One skilled in the art will appreciate thatthe leads, devices such as signal generators, systems and methodsdescribed herein can be practiced with embodiments other than thosedisclosed. The disclosed embodiments are presented for purposes ofillustration and not limitation. One will also understand thatcomponents of the leads depicted and described with regard the figuresand embodiments herein may be interchangeable.

What is claimed is:
 1. A method for detecting an analyte in a sample,comprising: contacting an analyte or an analyte and a tag-linked analytemolecule, a first recognition component, and an amplificationelement-linked second recognition component to generate a complexcomprising the first recognition component and the amplificationelement-linked second recognition component, wherein the firstrecognition component is immobilized relative to a surface of a thinfilm bulk acoustic resonator (TFBAR) and is configured to selectivelybind one or more of the analyte, the analyte molecule to which the tagis linked or the tag, or any one or more of these molecules bound to thesecond recognition component, and wherein the amplificationelement-linked second recognition component is configured to selectivelybind the analyte, the analyte molecule to which the tag is linked or thetag, or any one or more of these molecules bound to the firstrecognition component; contacting the linked amplification element withan amplification precursor under conditions to convert the amplificationprecursor into a molecule that adds mass at a surface of the TFBAR; andmeasuring mass added at the surface of the TFBAR.
 2. The method of claim1, wherein the analyte or the analyte and the tag-linked analyte arecontacted with the amplification element-linked second recognitioncomponent prior to contact with the first recognition componentimmobilized relative to the surface of the TFBAR.
 3. The method of claim1, wherein the analyte or the analyte and the tag-linked analyte arecontacted with the first recognition component prior to contact with theamplification element-linked second recognition component.
 4. The methodof claim 1, wherein the analyte or the tag-linked analyte, the firstrecognition component and the amplification element-linked secondrecognition component are contacted simultaneously.
 5. The method ofclaim 1, wherein measuring the mass added to our bound to the surface ofthe TFBAR comprises: coupling an input electrical signal to the TFBAR,the input electric signal having a frequency within a resonance band ofthe piezoelectric resonator, wherein the frequency is about 900 MHz orgreater; transmitting the input electrical signal through or across theTFBAR to generate an output electrical signal having a frequency;receiving the output electrical signal from the TFBAR; and determining achange in phase shift of the output electrical signal caused bydeposition of the precipitate on the surface of the TFBAR.
 6. The methodof claim 5, wherein the change in phase shift is a change in insertionor reflection coefficient phase shift.
 7. The method of claim 1, whereinmeasuring the mass added to our bound to the surface of the TFBARcomprises: actuating the TFBAR into an oscillating motion at a frequencyof about 900 MHz or greater; measuring one or more resonator outputsignals representing resonance characteristics of the oscillating motionof the TFBAR; and adjusting the actuation frequency of the sensingresonator to maintain a resonance point of the TFBAR.
 8. The method ofclaim 7, wherein the resonance point of the TFBAR is a point of maximumgroup delay.
 9. The method of claim 5, wherein the frequency is about1.8 GHz or greater.
 10. The method of claim 5, wherein the frequency isabout 2 GHz or greater.
 11. The method of claim 5, wherein the frequencyis from about 800 MHz to about 10 GHz.
 12. The method of claim 5,wherein the frequency is from about 2 GHz to about 2.5 GHz.
 13. Themethod of claim 1, wherein the amplification element is an enzyme andthe amplification precursor is a substrate, and wherein the enzyme isconfigured to convert the substrate to a precipitate.
 14. A system fordetecting an analyte in a sample, comprising: a thin film bulk acousticresonator (TFBAR) comprising a surface to which a first recognitioncomponent is immobilized, the first recognition component beingconfigured to selectively bind the analyte, an analyte molecule to whicha tag is linked, or a tag, or any one of these molecules to which anamplification element-linked second recognition component is bound, theTFBAR having a resonance frequency of 900 MHz or greater; one or morecontainers housing an amplification molecule, the amplificationelement-linked second recognition component, and optionally one or moreof the tag, and the analyte molecule; a fluid path from the one or morecontainers to the surface of the TFBAR to which the first recognitioncomponent is bound; actuation circuitry configured to drive the TFBAR inan oscillating motion; measurement circuitry arranged to be coupled tothe TFBAR and configured to measure one or more resonator output signalsrepresenting resonance characteristics of the oscillating motion of thesensing resonator; and a controller operatively coupled with theactuation and measurement circuitry.
 15. A kit for use with a device fordetecting an analyte in a sample, comprising: a thin film bulk acousticresonator (TFBAR) comprising a surface to which a first recognitioncomponent is immobilized, the first recognition component beingconfigured to selectively bind the analyte, an analyte molecule to whicha tag is linked, or a tag, or any one of these molecules to which anamplification element-linked second recognition component is bound, theTFBAR having a resonance frequency of 900 MHz or greater; and one ormore containers housing an amplification molecule, the amplificationelement-linked second recognition component, and optionally one or moreof the tag, and the analyte molecule.